Herpesviruses are a significant cause of disease in the human population. Following initial acute infection, herpesviruses establish latency and the viral DNA is maintained in cells. Murine gammaherpesvirus-68 (MHV-68) is used as a model to study the human gammaherpesviruses, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, as MHV-68 shares homology with and demonstrates similar immune responses to these infections. In this application we aim to examine a highly conserved viral enzyme, the MHV-68 ORF64 ubiquitin-specific protease (USP). We have generated mutant viruses with which to conduct these studies and have implemented assays to examine changes in viral and cellular gene expression and to quantitate viral load, infectious viral titer, the establishment of latency, reactivation, and immune responses following infection. Additionally, we have proposed a systematic proteomics approach to determine the substrate and interacting partners of the USP. Mutation of the USP in MHV-68 and other herpesviruses leads to a decrease in disease pathology;therefore, we hypothesize that this protease is essential in modulating virus-host interactions, perhaps playing a role in altering the cell cycle or immune responses to the virus. We additionally propose to analyze the contribution of the CDST cell response in controlling MHV-68 acute replication and establishment of latency. CDS T cells may undergo affinity maturation following viral infection;therefore, we propose to examine changes in T cell receptor (TCR) affinity for specific MHV-68 epitopes. Toward this end, we have discovered 19 novel MHV-68 MHC class I epitopes. We will examine the importance of these epitopes by generating virus mutants in which the epitopes are unable to be presented by MHC class I molecules. Generation of MHV-68 CDSTCR transgenic mouse lines will also help elucidate the function of the cytotoxic T lymphocyte response in controlling viral replication. PUBLIC HEALTH RELEVANCE: Elucidating the function of the highly conserved herpesviral USP is important in understanding the pathogenicity of MHV-68. Long-term control of viral infection and the development of a vaccine requires proper activation of the CDST cell response to class I MHC epitopes.